Supercritical fluid extraction (SFE) is a sample preparation technique used to extract analytes of interest from a sample, for example, fat from a food sample. Some type of sample preparation must be performed for a wide range of environmental, food, polymer, petroleum, pharmaceutical and other classes of samples due to the complex nature of the samples. Many of these samples are so complex that they cannot be directly analyzed by analytical techniques such as gas chromatography (GC) or liquid chromatography (LC). The complex samples must first go through a sample preparation step to perform a gross separation of the analytes of interest from the sample matrix in which they are contained, for example, the environmental pollutants from soil. After the sample preparation step, then just the analytes of interest are analyzed by the analytical technique such as gas chromatography (GC), liquid chromatography (LC), or supercritical fluid chromatography (SFC). Other analytical techniques could be used such as mass spectroscopy (MS) or nuclear magnetic resource (NMR).
The most popular sample preparation steps are Soxhlet extraction and liquid extraction. An alternative to these types of extraction techniques is supercritical fluid extraction (SFE). SFE offers a relatively rapid, simple and inexpensive technique to perform sample preparations. The basis of SFE is that a fluid, such as carbon dioxide, is held at a specific pressure, temperature and flowrate, which is above its critical temperature and pressure and thus is a supercritical fluid. The supercritical fluid is passed through the sample matrix containing the analytes of interest. This sample matrix is contained in an extraction vessel. The fluid diffuses into the pores of the matrix, solubilizes the analytes of interest, and then carries the analytes away from the matrix. The analytes are then collected by some device, so that the analytes can be analyzed by some further analytical technique, such as chromatography. The matrix (now without analytes) is left behind in the extraction vessel. Supercritical fluids have favorable diffusivities and viscosities providing for good mass transfer characteristics. Their solvent strength can be easily controlled by changing fluid pressure or temperature. These are but a few of the advantages of supercritical fluid extraction.
Typically, an SFE system is comprised of a pump which pumps the supercritical fluid to an extraction vessel where analytes are extracted from a sample matrix. The analytes are then transported to a collection device where the supercritical fluid is depressurized to ambient pressure and is vented. The analysis of the collected analytes can be either "off-line", that is, remote from the extraction and/or collection device, or "on-line", that is, fluidically connected to the extraction and/or collection device.
The primary supercritical fluid used in SFE is carbon dioxide due to its low pressure and temperature critical points (71 atm, 31.degree. C., respectively) and its ability to solubilize nonpolar or moderately polar analytes. When it is desired to extract a polar analyte, then it is well known in the art to employ a co-solvent with the carbon dioxide. These co-solvents are typically referred to as modifiers or entrainers and are typically a liquid organic solvent such as methanol, ethanol, propylene carbonate, acetone, tetrahydrofuran, fomic acid, etc. that are blended with the carbon dioxide in 1 to 80% by volume or mole percent to form a mixture that retains much of the diffusion characteristics of the pure carbon dioxide phase but that has a much higher polarity and thus is able to solubilize polar analytes and extract the polar analytes from the sample matrix.
Supercritical fluid extraction (SFE) has been utilized for some time in the extraction of fats and lipids in various matrices. Stahl, et al., E. Stahl, K. W. Quirin, D. Gerard. Verdichtete Gas zur Extraktion und Raffination. Springer-Verlag Heidelberg (1987)! used supercritical CO.sub.2 for the extraction and refining of oils seeds. Froning, et al. G. W. Froning, R. L. Wehling, S. L. Cuppett, M. M. Pierce, L. Niemann. D. Siekman, J. Food Sci. 55, 95 (1990)! have extracted cholesterol and other triglycerides from egg powder. King, et al. J. W. King, J. H. Johnson, J. P. Friedrich. J. Agric. Food Chem. 37, 951 (1989); J. W. King, J. H. Johnson, Fifth International Symposium on SFC and SFE, (Jan. 10-14, 1994)! used SFE on dehydrated foods and meats to extract free lipid fractions and total fat. Lembke and Engelhardt P. Lembke, H. Engelhardt, Fifth International SFE/SFC Symposium, (Jan. 10-14, 1994); H. Engelhardt, P. Lembke, Chromatographia, 34, 509 (1993)! recently used SFE for total fat determinations in cheese and meat sample matrices, using a pre-extraction acid treatment of the sample to enhance the accessibility of the lipids that were associated with carbohydrates and proteins in those sample matrices.
Recently, the accurate labeling requirements for fat in foods has been revised by federal agencies (FDA and USDA). This is a result of the Nutrition Labeling and Education Act which requires the labeling of total, saturated and unsaturated fats packaged in foods M. Clemmitt, Scientist, 8, (Nov. 1991)!. In addition, food manufacturers also require the consistent determination of fat for quality control purposes.
Presently, these determinations are performed by such techniques as Soxhlet (i.e., petroleum ether) extractions, the Roese-Gottlieb method (for milk products), or one of several acid hydrolysis steps followed by liquid solvent extraction. Attempts to eliminate the use of toxic, flammable solvents, to reduce conventional extraction times (i.e., 12-16 hours), and to have an independent monitor of fat levels has prompted food manufacturers to search for alternative methods. For example, near infrared spectroscopy has been used for fat determinations in food stuffs, but requires frequent calibrations and updating. SFE is practical alternative for fat determinations. By employing non-toxic, non-flammable solvents, namely supercritical carbon dioxide (CO.sub.2), SFE can be used, due to the physical properties of supercritical fluids, to yield precise and rapid fat extractions eliminating the need for liquid solvent disposal. Moreover, due to recent instrumental developments, especially in decompression control, SFE can be performed reliably and in a very straight forward, routine fashion. This kind of ease of operation is important, for example, for food manufacturing plant laboratories. For instance, supercritical fluid extraction has been utilized for the gravimetric determination of fats in snack foods (i.e. potato chips, tortilla chips, corn chips, popcorn). With supercritical carbon dioxide (CO.sub.2), fats can be extracted in less than one hour in a reproducible fashion, either manually or sequentially automated.